The Golgi-Localized Arabidopsis Endomembrane Protein12 Contains Both Endoplasmic Reticulum Export and Golgi Retention Signals at Its C Terminus

Abstract

Endomembrane proteins (EMPs), belonging to the evolutionarily conserved transmembrane nine superfamily in yeast and mammalian cells, are characterized by the presence of a large lumenal N terminus, nine transmembrane domains, and a short cytoplasmic tail. The Arabidopsis thaliana genome contains 12 EMP members (EMP1 to EMP12), but little is known about their protein subcellular localization and function. Here, we studied the subcellular localization and targeting mechanism of EMP12 in Arabidopsis and demonstrated that (1) both endogenous EMP12 (detected by EMP12 antibodies) and green fluorescent protein (GFP)-EMP12 fusion localized to the Golgi apparatus in transgenic Arabidopsis plants; (2) GFP fusion at the C terminus of EMP12 caused mislocalization of EMP12-GFP to reach post-Golgi compartments and vacuoles for degradation in Arabidopsis cells; (3) the EMP12 cytoplasmic tail contained dual sorting signals (i.e., an endoplasmic reticulum export motif and a Golgi retention signal that interacted with COPII and COPI subunits, respectively); and (4) the Golgi retention motif of EMP12 retained several post-Golgi membrane proteins within the Golgi apparatus in gain-of-function analysis. These sorting signals are highly conserved in all plant EMP isoforms and, thus, likely represent a general mechanism for EMP targeting in plant cells.

Dual roles of an Arabidopsis ESCRT component FREE1 in regulating vacuolar protein transport and autophagic degradation

“…To further gain insight into the effects of the free1 mutation at the ultrastructural level, we next performed transmission electron microscopic (TEM) analysis of ultrathin sections of free1 mutant seedlings processed by high pressure freezing/freezesubstitution (HPF/FS) (23). Consistent with the confocal observation in Fig.…”

“…The general procedures for TEM sample preparation, thin sectioning, and immunogold labeling were performed essentially as described previously (16,23). Root tips of 7-d-old wild-type and free1 mutant Arabidopsis plants were cut and immediately frozen in a highpressure freezer (EM PACT2, Leica), followed by subsequent freeze substitution in dry acetone containing 0.1% uranyl acetate at −85°C in an AFS freeze-substitution unit (Leica).…”

Activation of the Rab7 GTPase by the MON1-CCZ1 Complex Is Essential for PVC-to-Vacuole Trafficking and Plant Growth in Arabidopsis

“…Maintenance of Arabidopsis suspension-cultured cells and transient expression methods were described previously (Tse et al, 2004;Miao and Jiang, 2007;Gao et al, 2012;Zhuang et al, 2013). Confocal images were collected at 12 to 14 h or 36 to 48 h after transformation as indicated using an Olympus FV1000 system.…”

Sorting Motifs Involved in the Trafficking and Localization of the PIN1 Auxin Efflux Carrier

“…The general procedures for transmission electron microscopy sample preparation, thin sectioning, and immunogold labeling were performed essentially as described previously (Tse et al, 2004;Gao et al, 2012). Root tips of 4-d-old Arabidopsis seedlings expressing PIN1:GFP or PIN1:GFP-F165A were cut and immediately frozen in a high-pressure freezer (EM PACT2; Leica), followed by subsequent freeze substitution in dry acetone containing 0.1% uranyl acetate at 285°C in an AFS freeze substitution unit (Leica).…”